Secondary active transporters are a class of membrane proteins that utilize pre-existing molecular concentration gradients as an energy source for translocating another substrate, such as a nutrient or a neurotransmitter, against its concentration gradient. This process requires the protein to change conformations so as to expose a pathway to the substrate binding site(s) on one or other side of the membrane, in a cycle known as alternating access. Every organism expresses dozens of different secondary transporter proteins, and these exhibit a diverse set of architectures, albeit always with some form of internal structural symmetry. Unprecedented, ground-breaking insights have been garnered from three-dimensional structures obtained in the last decade. Nevertheless, a detailed understanding of the mechanism of each membrane transport protein requires knowledge of its structure in many more conformational states, including identification of the binding regions for the substrate or substrates. Moreover, those structures need to be placed into a context of dynamic ensembles on a thermodynamic landscape separated by kinetic barriers. Studies from our group over the last year have provided such insights into biomedically important transporters, with a focus on those in the brain and in gut microbes that are implicated in uptake of the neurotransmitter serotonin. Recent structural studies have revealed how the neuronal plasma membrane serotonin transporter SERT binds to certain inhibitors, such as paroxetine, which wedge the protein open, like a foot in a door. However, its mechanism of transport requires that the door can close when serotonin binds, and that this closure occur selectively. To understand the specific interactions involved in this process, we developed a structural model of SERT bound to all its required substrates, including serotonin, sodium, and chloride, and carried out molecular dynamics simulations in order to investigate quantitatively which interactions are likely to be required for the coupling mechanism (see ref. 1). These structural and modeling studies, carried out with the Ecker and Stary-Weinzinger labs (Vienna) provide testable hypotheses which we will investigate in future collaborative efforts. Only a small fraction of the serotonin in the body is actually found in the brain. In fact, over 80% of the serotonin in the body is present in the intestines, where it is required for signaling processes. Recent studies have shown that spore-forming microbiota contribute to regulating serotonin levels in the gut by promoting 5-HT biosynthesis in the host. However, whether serotonin plays a reciprocal role, namely in influencing the behavior of gut microbes is not known. Our collaborators in the Hsaio lab (UCLA), demonstrated that elevating levels of intestinal lumenal 5-HT by oral supplementation or genetic deficiency in the host 5-HT transporter (SERT) increases the relative abundance of spore-forming members of the gut microbiota in mice. After identifying a homologous protein to human SERT in Turicibacter sanguinis, we predicted its structure and identified some common features for serotonin uptake systems, leading to the hypothesis that this bacterium is able to take up serotonin and may be inhibited by selective-serotonin reuptake inhibitors. Indeed, the Hsaio lab found that T. sanguinis may import 5-HT through a mechanism that is inhibited by the selective 5-HT reuptake inhibitor fluoxetine. Moreover, the presence of 5-HT and fluoxetine influence the expression of sporulation factors and membrane transporters, which in turn appear to affect its ability to colonize the gastrointestinal tract. This work was published very recently (see ref. 2). In summary, our publications this year reflect ongoing efforts to utilize modeling in close collaboration with experimental laboratories, and drive understanding of the mechanism of secondary active transport related to neurotransmitter transmission, host-gut interactions, mechanisms of antidepressants, and to many other biomedically important mechanisms.